The present invention relates to a method for isolating and identifying a recombinant clone having a DNA segment therein coding for a polypeptide at least a short amino acid sequence of which is known. The present method is illustrated by its application to the production of clones containing a plasmid incorporating DNA coding for human histocompatibility antigens of the HLA-B region.
Recombinant DNA techniques are not quite well known. See, e.g., Morrow, "Recombinant DNA Techniques" in Methods In Enzymology, 68, 3-24 (Academic Press, 1979), and references cited therein, all of which are incorporated herein by reference. See also UK patent application Nos. GB2,007,675A; 2,007,676A and 2,008,123A, the disclosures of which are also incorporated herein by reference. Genes coding for various polypeptides may be cloned by incorporating a DNA fragment coding for the polypeptide in a recombinant DNA vehicle, e.g., a bacterial or viral plasmid, and transforming a suitable host, typically an Escherichia coli (E. coli) cell line, and isolating clones incorporating the recombinant plasmids. Such clones may be gown and used to produce the desired polypeptide on a large scale.
DNA for cloning may be obtained in at least three ways. DNA extracted from cellular material may be used, either as such or after excision of various portions. Alternatively, DNA can be synthesized chemically, provided the nucleotide sequence is known or the amino acid sequence of the desired polypeptide is known.
A third method of producing DNA for cloning is reverse transcriptase catalyzed synthesis in the presence of mRNA. However, known techniques for isolating and identifying cDNA produced from RNA by reverse transcription are inadequate for cloning cDNAs for which the corresponding mRNA constitutes a very minor proportion of the total mRNA from a cell type.
The use of oligonucleotide primers to detect yeast cytochrome C mRNA and gastrin mRNA has been reported (Szostak et al., Nature, 265, 63 (1977); Noyes et al., Proc. Natl. Acad. Sci. USA, 76, 1770 (1979)). The use of dideoxynucleoside triphosphates (ddNTPs) and arabinosyl triphosphates for sequencing DNA (Sanger, et al., Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)) and for sequencing RNA (Zimmeren et al., Proc. Natl. Acad. Sci. USA, 75, 4257 (1978)) is known.
The histocompatibility region (HLA) in humans is a complex gene family located on chromosome 6. The genes in this region are related to immune responses, and tend to be highly polymorphic, reflecting the highly individual immune response of humans. The best studied and most relevant loci within the HLA region are the HLA-A, HLA-B, HLA-C, HLA-D and HLA-DRW loci. See Perkins. "The Human Major Histocompatibility Complex (MHC)", in Basic And Clinical Immunology, 2d Edition, 165-174 (Lange Medical Publ., 1978) for further details regarding this complex.
Antigens coded for by the HLA loci are primarily proteins located on the cell's exterior surface, and allow the cells to recognize self from non-self. At a cellular level, these antigens are involved in the recognition of host from foreign cells, and enable a population of lymphocytes called helpers T cells to be activated and participate in the activation of macrophage, B cells and killer T cells. In attempts to graft tissue from a different donor, a mismatch of any of these HLA loci results in a graft/host reaction which ultimately can lead to the rejection of mismatched organs by patients.
The proteins coded for by the HLA region comprise only about 0.05 to 0.1% of the total cellular proteins and about 1% of the membrane associated protein of lymphoblastoid cell lines. The antigens of the HLA-A, HLA-B and HLA-C regions tend to co-purify and are therefore difficult to isolate. In addition, isolation of these antigens from human cell lines would be expensive. Consequently, the separation of individual genes and production of specific antigens by conventional procedures is fraught with difficulty.
Other genes of biological interest are also difficult to isolate, for many of the reasons that hold for HLA antigens. Even where full or partial amino acid sequences are available for the corresponding proteins, a low proportion of mRNA corresponding to the gene still make isolation and characterization difficult.
A need therefore continues to exist for a method for cloning genes for polypeptides whose corresponding mRNA is a minor fraction of the total mRNA in an mRNA mixture, especially a method sensitive to mRNA present in the mixture below the level of 2 mol%, and even below 0.01 mol%.